ORIGINAL PAPER

In vitro propagation of four threatened Paphiopedilum species(Orchidaceae)

Bo Long • Alex X. Niemiera • Zhi-ying Cheng •

Chun-lin Long

Received: 27 March 2009 / Accepted: 11 January 2010 / Published online: 2 February 2010

� Springer Science+Business Media B.V. 2010

Abstract The effects of seed maturity, media type, carbon

source, and organic nutrient additives on seed germination,

protocorm development, and plant growth of Paphiopedi-

lum villosum var. densissimum Z. J. Liu et S. C. Chen were

investigated. Micropropagation frequency was enhanced

through the use of 200-day-old seed, Knudson C (KC)

medium, and the presence of both glucose and coconut milk

in the medium. The effects of various plant growth regula-

tors on the frequency of shoot organogenesis in four

Paphiopedilum species were also investigated. Explants of

P. villosum var. densissimum and P. insigne (Lindl.) Stein

incubated in the presence of 5 mg l-1 6-benzyladenine (BA)

with 0.5 mg l-1 a-naphthalene acetic acid (NAA) and

0.2 mg l-1 BA with 0.1 mg l-1 NAA, respectively, showed

a twofold increase in the frequency of shoot organogenesis.

For explants of P. bellatulum (Rchb. f.) Stein and P. arme-

niacum S. C. Chen et F. Y. Liu, the combination of

5.5 mg l-1 BA with 0.5 mg l-1 NAA and 4 mg l-1 BA with

0.1 mg l-1 NAA, respectively, resulted in the highest fre-

quencies of shoot organogenesis.

Keywords Micropropagation � Orchid �Seed germination � Shoot proliferation

Abbreviations

2,4-D 2,4-Dichlorophenoxyacetic acid

AC Activated charcoal

BA 6-Benzyladenine

IAA Indole-3-acetic acid

IBA Indole-3-butyric acid

KT Kinetin

LH Lactalbumin hydrolysate

NAA a-Naphthalene acetic acid

TDZ 1-Phenyl-3-(1, 2, 3-thiadiazol-5-yl)-urea

Introduction

Paphiopedilum spp. (Orchidaceae), commonly known as

Lady’s slipper orchids, are often marketed as attractive and

desirable container-grown plants. A few species are

regarded as endangered or even extinct in the wild due to

over-collection from natural areas and large-scale illegal

trade. Paphiopedilum spp. have been designated as

endangered by the Convention on International Trade in

Endangered Species of Wild Fauna and Flora (CITES;

CITES Appendices 2008). In a natural setting, Paphio-

pedilum seed germinates relatively slowly due to the

absence of an endosperm. In P. armeniacum, the interval

between seed germination and tiller production is about 4

years (Liu et al. 2006), although some orchid hybrids and

tropical orchids initiate tiller production within 6 months

B. Long � Z. Cheng � C. Long (&)

Kunming Institute of Botany, Chinese Academy of Sciences,

650204 Kunming, China

e-mail: long@mail.kib.ac.cn; chunlinlong@hotmail.com

B. Long

e-mail: longbo@mail.kib.ac.cn

B. Long

Graduate School, Chinese Academy of Sciences, 100039

Beijing, China

A. X. Niemiera

Department of Horticulture, Virginia Polytechnic

and State University, Blacksburg, VA 24061, USA

e-mail: niemiera@vt.edu

C. Long

College of Life and Environmental Sciences,

Minzu University of China, 100081 Beijing, China

123

Plant Cell Tiss Organ Cult (2010) 101:151–162

DOI 10.1007/s11240-010-9672-1

of germination and tiller yearly (Chen and Tsi 2003).

P. villosum (Lindl. ex Hook.) Stein and P. barbigerum

T. Tang et F. T. Wang both show low frequencies of fruit

set, with average fruit set of 8 and 26%, respectively

(Banziger 1996; Shi et al. 2008). Although Paphiopedilum

spp. produce abundant seeds, germination levels in nature

are low and, consequently, Paphiopedilum spp. are rela-

tively rare in the wild (Arditti and Ernst 1993).

In general, the growth and development of in vitro-

grown plants depend on factors such as macro- and micro-

element composition, carbon source, and plant growth

regulator (PGR; Murashige and Skoog 1962; Xiong and

Wu 2003). Although sucrose is the most utilized carbon

source for plant tissue culture, other sugars, such as glu-

cose, fructose, maltose, and mannitol, are also effective (Li

2002). The frequencies of in vitro seed germination of

P. insigne var. sanderae (Rchb. f.) Stein and P. armenia-

cum are also influenced by seed maturity and organic

nutrient additives, such as banana, potato, and coconut

juice (Nagashima 1982; Pierik et al. 1988; Ding et al. 2004;

Lee 1998, 2007). Asymbiotic seed germination of fully

mature orchid seeds is often difficult (Lee et al. 2006), with

the frequency ranging from 35 to 68% in 12 Paphiopedi-

lum spp. (Lee 1998; Tay et al. 1988; Chen et al. 2004b;

Zeng et al. 2006; Lee 2007). In contrast, the germination

frequency is 97 and 100% for P. ciliolare (Rchb. f.) Stein

and P. callosum (Rchb. f.) Stein, respectively (Stimart and

Ascher 1981; Pierik et al. 1988).

There are relatively few published reports on the

micropropagation of Paphiopedilum spp. using tissue cul-

ture technology, primarily due to the difficulty in main-

taining explants in culture. P. delenatii has been induced to

form shoot buds by wounding and liquid culture of nodal

segments (Nhut et al. 2005) with a regeneration frequency

of 75% (Nhut et al. 2007). Efforts to establish long-term

Paphiopedilum callus cultures from stem segments have

been relatively unsuccessful. However, a few Paphioped-

ilum hybrids have been successfully propagated from seed-

derived protocorms, callus, shoot, leaf, and nodal tissues

(Stewart and Button 1975; Lin et al. 2000; Chen et al.

2004a; Huang et al. 2001). It is likely that Paphiopedilum

hybrids are easier to micropropagate than native species.

Consequently, the propagation of Paphiopedilum spp. is

mainly achieved by asymbiotic seed germination due to the

limited success of tissue culture protocols. Given the

environmental status of Paphiopedilum spp., its high

desirability in commercial horticultural markets, and the

need to save several Paphiopedilum species from extinc-

tion, it is essential that an efficient tissue culture micro-

propagation method for Paphiopedilum be established. The

existence of a rapid and large-scale in vitro propagation

protocol would also assist in restoring Paphiopedilum spp.

to native environments. Tissue culture-produced plantlets

can also be used in ex situ preservation, which is an

important approach to plant germplasm preservation, par-

ticularly in terms of pest-free cultures, high propagation

frequency, and gene stability.

The in vitro propagation of P. villosum var. densissimum

and shoot multiplication of P. bellatulum, P. insigne, and

P. armeniacum have not been documented in the literature.

In the study reported here, we carried out a series of

experiments aimed at establishing an efficient microprop-

agation system for native Chinese Paphiopedilum spp.,

including P. villosum var. densissimum, P. bellatulum,

P. insigne, and P. armeniacum. In particular, the influence

of seed maturity, medium composition, sugar type, and

organic nutrient additives on in vitro seed germination and

plant growth has been investigated. The effects of combi-

nations of cytokinins and auxins on in vitro callus induc-

tion and shoot proliferation were also investigated.

Materials and methods

Plant material

Paphiopedilum var. densissimum, P. insigne, P. bellatulum,

and P. armeniacum were collected from wild populations

in Wenshan County of Yunnan Province (southwestern

China). The plants were transplanted into containers and

grown in the greenhouse at the Kunming Institute of Bot-

any in Kunming, China.

Basal culture media and general culture conditions

The basal culture medium consisted of quarter-strength MS

(Murashige and Skoog 1962) micro- and macro-elements,

100 mg l-1 myoinositol, 1 g l-1 niacin, 1 g l-1 pyridoxine

HCl, 1 g l-1 thiamine HCl, and 6 g l-1 agar (plant tissue

culture tested; Sanland Int, Tokyo, Japan). The pH of the

medium was adjusted to 5.8 with 1 N KOH or HCl. Prior to

use, the medium was autoclaved for 20 min at 121�C. All

other experimental media treatments are described

separately.

For each treatment in different media, either 100 seeds or

three explants were used per vessel, and the treatments were

tested in triplicate. Seeds or explants were placed in the dark

for a period of either 2 or 4 weeks and then transferred to

light conditions under a 16/8-h (light/dark) photoperiod with

light provided by 36-W cool-white fluorescent bulbs

(36 lmol m-2 s-1) at 22 ± 2�C for a period of 3 months.

Seed germination and protocorm growth experiments

Flowers of plants from all four species were hand-polli-

nated. The time from hand pollination to inoculation

152 Plant Cell Tiss Organ Cult (2010) 101:151–162

123

treatment (placement on medium) varied in the different

experiments. Seed capsules were collected and sterilized

with 75% (v/v) ethanol for 30 s and then dipped into 0.1%

HgCl2 for 15–20 min followed by a thorough rinsing in

sterile distilled water. Approximately 300–400 extracted

seeds were placed on the basal medium amended with

20 g l-1 sucrose, 1 g l-1 activated charcoal (AC), and

100 ml l-1 coconut milk (liquid endosperm of freshly

harvested Cocos sp.) in 200-ml glass vessels (height

8 cm 9 width 7 cm) sealed with a plastic cap.

P. villosum var. densissimum seed maturity experiment

To determine the influence of seed maturity on seed ger-

mination, P. villosum var. densissimum seed capsules were

collected at 120, 130, 140, 150, 160, 170, 180, 190, 200, or

300 days after pollination (DAP). At each collection date,

seeds were placed on the basal medium supplemented with

100 ml l-1 coconut milk, 20 g l-1 sucrose, and 1 g l-1AC.

The germination frequency of about 100 seeds at 20, 40,

60, or 80 days after inoculation (placement on amended

medium) was determined by microscopic observation

(Axioskop 40; Zeiss, Gottingen, Germany). Germination

was verified by the appearance of a white protocorm and

was expressed as the frequency of all seeds sown. Proto-

corms turned green after approximately 100 days in cul-

ture, were re-measured, and then used in the plant growth

experiments. Plantlet leaf size was measured after 200

days.

P. villosum var. densissimum media composition

experiments

To determine the effect of media composition on germi-

nation and growth, 300-DAP seeds were placed on one of

the following media: Knudson (KC; 1946), Vacin and

Went (VW; 1949), quarter-strength MS (basal culture

medium) or half-strength MS. Each of these media were

supplemented with 100 ml l-1 coconut milk, 20 g l-1

sucrose, and 1 g l-1 AC. Germination frequency and

growth were measured as described above.

P. villosum var. densissimum media supplement

experiments: carbon source

To determine the effect of carbon source on seed germi-

nation, 300-DAP seeds were placed on MS basal culture

medium amended with 100 ml l-1 coconut milk and

1 g l-1 AC. This medium was then amended with 20 g l-1

sucrose, glucose, maltose, or mannitol. Germination fre-

quency and growth were measured as described above.

P. villosum var. densissimum media supplement

experiments: organic nutrient additives

To determine the effect of organic nutrient additives on

seed germination, 300-DAP seeds were placed on MS basal

culture medium amended with 20 g l-1 sucrose and

1 g l-1 AC and then amended with 100 g l-1 potato mash,

100 g l-1 apple mash, 100 g l-1 chayote mash, 4 g l-1

lactalbumin hydrolysate (LH), or 100 ml l-1 coconut milk.

The source of these additives were fresh potato, apple,

chayote, Solanum tuberosum L., Malus domestica Borkh.

‘Red Fuji’, Sechium edule (Jacq.) Swartz, respectively, that

were skinned and homogenized in a blender. Germination

frequency and growth measurements were measured as

described above.

Experiments testing the effect of PGRs on callus

formation

To determine the influence of PGRs on callus formation of

the four species, protocorms and leaf fragments (from the

seed germination and protocorm growth experiments) were

cultured in media amended with various PGR combina-

tions (described below). There were at least 30 protocorms

of each of the four species per container. Leaf fragments

from aseptic plantlets of all species were cut into 0.5-, 1.0-,

1.5-, or 2.0-cm2 pieces. Each of the 200-ml containers

contained four fragments (0.5, 1.0, 1.5, and 2.0 cm2,

respectively). The media treatments consisted of various

combinations of three cytokinins and two auxins: (1)

0.1 mg l-1 kinetin (KT) in combination with 1.0, 2.0, 4.0,

or 8.0 mg l-1 2, 4-dichlorophenoxyacetic acid (2,4-D); (2)

0.02, 0.05, 0.1, or 0.5 mg l-1 1-phenyl-3-(1, 2, 3-thia-

diazol-5-yl)-urea (TDZ) in combination with 1.0, 2.0, 4.0,

or 8.0 mg l-1 2,4-D; (3) 1.0, 2.0, 4.0, or 8.0 mg l-1 indole-

3-butyric acid (IBA) in combination with 0.1, 0.5, or

1.0 mg l-1 a-naphthalene acetic acid (NAA). The MS

basal culture medium was amended with 100 ml l-1

coconut milk, 20 g l-1 sucrose, and 1 g l-1 AC. The fre-

quency of callus induction and browning were recorded

after 3 months.

Experiments testing the effect of PGRs on shoot

multiplication

Protocorms (all four species; from seed germination and

protocorm growth experiments) were transferred to new

200-ml containers (three plantlets per vessel) and cultured

for 1 year to become 3- to 5-cm plantlets; these plantlets

were used as a source of shoot multiplication material. In

addition, 3-cm P. armeniacum rhizome pieces, each with a

node, were taken from the aseptic plantlets derived in the

seed germination experiment and also used as shoot

Plant Cell Tiss Organ Cult (2010) 101:151–162 153

123

multiplication material. The PGR treatments (Table 3)

were: (1) the cytokinin TDZ (0.01, 0.02, 0.05, 0.1 and

0.2 mg l-1) in factorial combination with the auxin NAA

(0.1 and 0.5 mg l-1); (2) various combinations of the

cytokinin BA (range: 0.2–6.0 mg l-1) with the auxin NAA

( 0.1 and 1.0 mg l-1). The basal medium was amended with

100 g l-1 potato mash, and 20 g l-1 glucose. The number of

new shoots and shoot length were recorded after 3 months.

After 2 months, seedlings in uncapped containers were

maintained under culture room conditions at 70% relative

humidity. After a 3-day acclimation period (gradual tran-

sition from culture room conditions to a high-humidity

greenhouse environment), the plantlets were removed from

the containers and the roots washed in tap water to remove

residual agar. The plantlets were then transplanted into

10-cm-diameter pots with a peat moss substrate and grown

in the greenhouse. The survival rate of the plantlets after

1 month in the greenhouse was recorded.

Statistical analyses

Analysis of variance (ANOVA) was performed with the

SPSS ver. 16.0 statistical package (SPSS, Chicago, IL) and

mean separation with the LSD test. A significance level of

P \ 0.05 was applied. Figures were constructed using

Sigmaplot 10.0 (Systat Software Richmond, CA).

Results

Seed germination and protocorm growth experiments

P. villosum var. densissimum seed: maturity experiment

A minimum of 150 DAP was required for the seeds to

germinate (Table 1; Fig. 4a). The highest germination

frequencies, 31 and 13%, were obtained for 200-day-old

seeds inoculated (placed on medium) for 80 and 40 days,

respectively. Germination frequencies for 300-day-old

seeds were lower than those for 200-day-old seeds, but in

both the germination rate 80 days following inoculation

(13 vs. 31%, respectively) was higher than that 40 days

following inoculation (0 vs. 13%, respectively; Table 1).

P. villosum var. densissimum media composition

experiment

The highest germination frequency of P. villosum var.

densissimum, 56%, was obtained on KC medium 80 days

after inoculation (Fig. 1). The germination frequency rate

in the VW treatment (37%) was lower than that in the KC

treatment but higher than that in the quarter- and half-

strength MS treatments (21 and 14%, respectively). The

protocorms were larger on the VW medium (2.6 mm2) than

on the one-half-strength MS medium after 100 days of

culture, while protocorm sizes in the quarter-strength MS

and KC treatments were the same as those in the VW

treatment (Figs. 1, 4b). Rhizoids were observed on VW

medium after 100 days (data not shown).

P. villosum var. densissimum media supplements

experiments: carbon source

The frequency of P. villosum var. densissimum seed ger-

mination on the glucose-amended medium (19%) was

higher than that on media containing other carbon sources

after 100 days of culture (Fig. 2). The germination fre-

quency was lowest for the mannitol treatment (0.08%).

Leaf length and width were greater in the glucose treatment

than in the mannitol treatment, but they were the same in

the sucrose, glucose, and maltose treatments.

P. villosum var. densissimum media supplements

experiments: organic nutrient additives

Seed germination frequency was highest in the coconut

milk treatment (10%; Fig. 3). Germination frequencies in

the apple and potato treatments were less than 3%, while

those in the chayote and LH treatments were 0%. Leaf

length was lowest in the LH treatment, and lengths and

widths were the same for all other treatments, with the

exception of leaf width in the LH treatment.

Table 1 Effect of seed maturity on in vitro seed germination of Paphiopedilum villosum var. dentissimum

Inoculation timea (days) Frequency of in vitro germination (%)

120 DAP 130 DAP 140 DAP 150 DAP 160 DAP 170 DAP 180 DAP 190 DAP 200 DAP 300 DAP

40 0 b 0 b 0 b 0 b 0 b 0 b 0 b 0.43 b 13 a 0 b

80 0 c 0 c 0 c 0.12 c 0.14 c 0.74 c 0.9 c 0.71 c 31.33 a 13.33 b

Data are given as the mean in vitro germination frequency of seed of P. villosum var. densissimum collected from fruit harvested between 120

and 300 days after pollination (DAP). Each mean is based on microscopic observation. Means followed by different lower-case letters are

significantly different at P \ 0.05 level by the LSD testa Duration of time between placement of medium (plating) and germination

154 Plant Cell Tiss Organ Cult (2010) 101:151–162

123

The effect of PGRs on callus formation

Few seed-derived protocorms induced callus (data not

shown; Fig. 4c, d). All leaf pieces taken from 1.5-year-old

plantlets (cultured in containers; Fig. 4e) as callus induction

material turned brown and ultimately died. The combination

of 0.5 mg l-1 NAA with 2.0 mg l-1 BA resulted in leaves

that remained green (no browning, no callus) during the

Medium

Fre

quen

cy o

f ger

min

atio

n %

¡r/ p

roto

corm

siz

e m

m2

0

10

20

30

40

50

60

70Frequency of germination after 80days following inoculationPromcorm size after 100days following inoculation

C

A

B

C

ab ab ab

1/4MS KC VW 1/2MS

Fig. 1 Effect of medium composition on seed germination and

protocorm size of in vitro Paphiopedilum villosum var. dentissimum.Mean in vitro seed germination and protocorm size of P. villosum var.

densissimum in relation to composition of the medium. Data were

recorded 3 months following culture initiation. Each mean is based on

a visual observation. a Data were square-root transformed prior to

analysis of variance (ANOVA) and converted to the original scale for

demonstration in the figure. b Means with different letters within a

column are significantly different at P \ 0.05 level by the LSD test.

Bars: Standard errors. KC Knudson medium, VW Vacin and Went

medium, MS Murashige and Skoog medium (1/4, 1/2 quarter- and

half-strength, respectively). Details on the various media are given in

the text

Carbon sourceSucrose Glucose Maltose Mannitol

Freq

uenc

y of

ger

mia

ntio

n (

%)

/ Lea

f len

gth

(mm

)

0

5

10

15

20

25

Frequency of germinationLeaf lengthLeaf width

A

B

C

D

aba

ab

b

a a

ab

b

/ Lea

f wid

th (

mm

)

Fig. 2 Effect of carbon source

on mean in vitro seed

germination and leaf size of in

vitro Paphiopedilum villosumvar. dentissimum. Data were

recorded 3 months following

culture initiation. Each mean is

based on a visual observation. aData were expressed as

germination frequency; leaf

length was square-root and

reciprocal square-root

transformed prior to ANOVA

and converted to the original

scale for demonstration in the

figure. b Means with different

letters within a column are

significantly different at the

P \ 0.05 level by the LSD test.

Bars: Standard errors

Plant Cell Tiss Organ Cult (2010) 101:151–162 155

123

0

5

10

15

20

25

Frequency of germinationLeaf lengthLeaf width

Potato Apple Chayote LH Coconut

Organic nutrient additives

Fre

quen

cy o

f ger

min

atio

n (

%)

/ Le

af le

ngth

(m

m)

B

B

C C

A

a

a

a

b

a

a aab

bab

/ Le

af w

idth

(m

m)

Fig. 3 Effect of organic

nutrient additives on mean seed

germination and leaf size of

P. villosum var. dentissimum in

vitro. Data were recorded

3 months following culture

initiation. each mean is based on

a visual observation. Means

with different letters within a

column are significantly

different at the P \ 0.05 level

by the LSD test. LHLactalbumin hydrolysate. Bars:

Standard errors

Fig. 4 In vitro propagation process of Paphiopedilum spp. aGermination of 300-DAP (day after pollination) seed of P. villosumvar. densissimum: swollen embryo and ruptured testa (Bar: 0.1 mm).

b Protocorm growth (Bar: 1 mm), c Callus mass developed from a

protocorm of P. bellatulum. d Shoot cluster formed from callus of P.bellatulum. e 1.5-year-old P. insigne in vitro seedling with roots. f–I

Shoot multiplication from seedlings of P. villosum var. densissimum(f), P. insigne (g), P. bellatulum (h), and P. armeniacum (I).

j P. insigne root tubercles. k–m Transplanted seedlings of P. villosumvar. densissimum (k), P. insigne (l), and P. bellatulum (m) in a pot

(Bar: 1 cm)

156 Plant Cell Tiss Organ Cult (2010) 101:151–162

123

3-month culture period, but after subculture, the leaves

gradually turned brown (Table 2). A number of PGR com-

binations reduced the amount of browning, such as in the

case of P. insigne treated with 2,4-D (at 2.0, 4.0, and

8.0 mg l-1) in combination with KT (0.1 mg l-1) and TDZ

(0.02, 0.05 mg l-1; Table 2). Explant size also affected the

browning phenomenon; 1.5-cm2 leaf pieces had less

browning than all other sizes, and 2-cm2 pieces showed the

most browning (data not shown).

The effect of PGRs on shoot multiplication

The length and number of shoots of the four Paphiopedi-

lum spp. was influenced by the concentrations and com-

binations of cytokinins and auxins contained in the medium

(Table 3; Fig. 4f-I). In a 3-month period, three new

plantlets were produced (per three plants) by: (1) P.

villosum var. densissimum for the combinations of

3.0 mg l-1 BA with 1.0 mg l-1 NAA, 5.0 mg l-1 BA with

0.5 mg l-1 NAA, and 6.0 mg l -1 BA with 0.1 mg l-1

NAA; (2) P. insigne for the combinations of 0.2 mg l-1

BA with 0.1 mg l-1 NAA and 1.0 mg l-1 BA with

0.5 mg l-1 NAA. For P. bellatulum, the combination

5.5 mg l-1BA with 0.5 mg l-1 NAA induced maximum

shoot organogenesis (0.33; one new plantlet per three

plants) and produced the longest shoots (0.6 cm); this same

result was obtained with the 1.0 mg l-1 BA and 0.5 mg l-1

NAA treatment. Organogenesis occurred im all PGR

combinations for P. armeniacum; for this species, shoot

number was highest in the 4.0 mg l-1 BA and 0.1 mg l-1

NAA treatment (Table 3).

After 2 months, plantlets with three or more 3- to 6-cm-

long roots and four to five leaves were transferred to pots

with a peat moss substrate (Fig. 4k–m). Although the

survival frequency of these greenhouse-grown rooted

plants was about 60%, the seedlings grew very slowly.

Discussion

Seed germination and protocorm growth in vitro

Seed maturity significantly affected the germination fre-

quency of P. villosum var. densissimum (Table 1). The

germination frequency of 200-DAP seeds was highest on

the basal medium (inoculation); seed coat formation started

about 200 DAP. Seeds\200-DAP turned brown soon after

inoculation, indicating that the embryo was seemingly too

underdeveloped to absorb nutrients from the medium. In

contrast, the frequency of germination for 300-DAP seed

was lower than that of 200-DAP seed, likely because the

seed coat had already formed (microscopic observation),

possibly attenuating nutrient uptake. A similar effect of

seed maturity on the germination of Paphiopedilum seed

has been shown by Nagashima (1982) and Ding et al.

(2004). Ren and Wang (1987) found that if a P. godefroyae

(Godef.) Stein embryo had 10–40 cells at 100 ± 5 DAP,

then suspensor and endosperm nuclei degenerated at

120 ± 5 DAP; however, at 200 ± 10 DAP, the globular

embryo of mature seed had 120–140 cells, and there was a

single cell layer in the seed coat. Lee (1998) reported

optimal seed germination of P. delenatii Guillanmin at 150

DAP (68%), with the germination frequency decreasing

Table 2 Effect of cytokinins in combination with auxins on brown-

ing of leaf fragments

Cytokinins (mg l-1) Auxin (mg l-1) Browninga (%)

2,4-D NAA

KT

0.1 1 0 100

0.1 2 0 66.7

0.1 4 0 57.1

0.1 8 0 42.9

TDZ

0.02 1 0 28.6

0.02 2 0 71.4

0.05 4 0 33.3

0.05 8 0 71.4

0.1 1 0 85.7

0.1 2 0 100

0.5 4 0 100

0.5 8 0 100

BA

1 0 0.1 100

2 0 0.1 100

4 0 0.1 100

8 0 0.1 100

1 0 0.5 100

2 0 0.5 0

4 0 0.5 100

8 0 0.5 100

1 0 1 100

2 0 1 100

4 0 1 100

8 0 1 100

KT Kinetin, TDZ 1-phenyl-3-(1, 2, 3-thiadiazol-5-yl)-urea, BA 6-ben-

zyladenine, 2,4-D 2,4-dichlorophenoxyacetic acid, NAA a-naphthalene

acetic acida Mean browning of Paphiopedilum insigne leaf fragments following

culture in medium containing various combinations and concentra-

tions of cytokinins and auxins. Browning was recorded after 3 months

of culture. Each mean is based on a visual observation

Plant Cell Tiss Organ Cult (2010) 101:151–162 157

123

Ta

ble

3E

ffec

to

fcy

tok

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sin

com

bin

atio

nw

ith

aux

ins

on

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enes

isd

uri

ng

the

cult

ure

of

exp

lan

ts

Cy

tok

inin

sA

ux

inP

.vi

llo

sum

var

.d

ensi

ssim

um

P.

insi

gn

eP

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ella

tulu

mP

.a

rmen

iacu

m

TD

Z

(mg

l-1)

BA

(mg

l-1)

NA

A

(mg

l-1)

Sh

oo

t/ex

pla

nt

(no

.)

Sh

oo

tle

ng

th

(cm

)

Sh

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t/ex

pla

nt

(no

.)

Sh

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tle

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th

(cm

)

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tub

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(cm

3)

Sh

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Sh

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tle

ng

th

(cm

)

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(no

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tle

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(cm

)

0.0

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00

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0.6

71

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.66

b

0.0

50

0.1

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1.0

81

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1.1

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.93

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0.2

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0.1

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1.0

70

.73

b

0.0

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0.5

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0.7

20

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0.0

20

0.5

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.33

0.8

0a

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0.4

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.33

0.5

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0.9

60

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b

0.0

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0.5

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1.1

0a

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0.7

3b

0.1

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0.5

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0.3

3c

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.98

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4b

0.2

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0.5

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1.1

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.90

b

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.20

0.0

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0.9

7a

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0.3

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1.0

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0.2

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158 Plant Cell Tiss Organ Cult (2010) 101:151–162

123

drastically at 210 DAP when the seeds were fully mature.

At 150 DAP, the cuticular layer was not fully formed and

suspensor cells were vacuolated, thereby enabling func-

tional nutrition uptake (Lee 1998). Our results as well as

those of Ren and Wang (1987), Lee (1998, 2007), and Ding

et al. (2004) show that the influence of seed maturity on

germination is species-specific. Orchid seed germination is

relatively low due to the fact that an endosperm fails to

develop during seed development (Lee et al. 2006); in

addition, a thin seed coat may not sufficiently protect the

embryo from desiccation. The formation of a prominent

cuticle on the embryo surface may ensure moisture reten-

tion by embryo cells as well as offer physical protection.

However, the cuticle lowers the success rate of in vitro

orchid seed germination (Lee et al. 2006).

We found that the relatively low mineral salt and

inorganic N concentrations in KC and VW medium pro-

moted seed germination of P. villosum var. densissimum

(mineral salt concentrations: KC, 13.42 mM, WV,

16.3 mM; inorganic N concentrations: KC, 12.25 mM,

VW, 3.78 mM; Fig. 1). Germination frequency was high-

est on the medium amended with KC; this result is similar

to that reported by Johnson and Kane (2007) working with

Vanda hybrids (orchid). In contrast, Bhaskar and Rajeevan

(1996) reported a higher frequency of germination for

Vanda ‘John Club’ on half-strength MS than on KC, while

Roy and Banerjee (2002), working with Vanda tessellata

(Roxb.) Hook. ex Don, obtained similar germination rates

on half-strength MS and KC. The stimulative effect of the

KC medium on germination frequency may be related to

the fact that KC has a relatively high calcium concentra-

tion (4.23 mM) compared to VW (1.94 mM), and quarter-

and half-strength MS (0.75 and 1.5 mM, respectively). The

supply of calcium significantly influences plant growth and

cell karyokinesis. Larger protocorms were obtained on VW

than on half-strength MS (Fig. 1), which may be related to

the relatively high phosphate concentration (4.74 mM) of

VW compared to quarter- and half-strength MS (0.32 and

0.62 mM, respectively) and KC (1.84 mM). Phosphate

uptake by rhizoids (which emerged after embryo swelling

and testa breakage) may have stimulated protocorm

growth. This response is similar to other observations on

the orchid Bletia purpurea (Lam.) DC (Dutra et al. 2008).

The germination frequency of P. villosum var. densiss-

imum was highest on the glucose-amended medium and

was almost twofold higher than that on the sucrose-amen-

ded medium (Fig. 2), which is consistent with the obser-

vations of Cao and Jia (2003), Xie (2003), Cuenca and

Vieitez (2000), and Abdoulaye and Mark (2006). Maltose

has been reported as being an effective sugar source for

Oncidium ‘Gower Ramsey’ plantlet growth (Jheng et al.

2006). However, our findings showed that maltose was less

effective in promoting germination in Paphiopedilum,Ta

ble

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Plant Cell Tiss Organ Cult (2010) 101:151–162 159

123

which is consistent with the observations of Huang et al.

(2001), Shen and Xu (1997), Oldach et al. (2001), Cao and

Jia (2003), and Abdoulaye and Mark (2006).

Potato is generally used more frequently than apple as a

natural substance in tissue culture work. Our results show

that the germination frequency and leaf size of P. villosum

var. densissimum in the apple and chayote treatments were

the same as those found in the potato treatment (Fig. 3).

Potato is most likely used for its easy storage and inex-

pensiveness. Because components of natural addenda differ

depending on variety, production area and methods, and

fruit maturity, our results serve mainly as an indication of

the relative influence of different natural sources, espe-

cially in terms of Paphiopedilum germination.

Callus induction

Our results demonstrate that, irregardless of the callus

induction media treatment and size of the in vitro leaf

fragment, the inducement of callus formation was generally

unsuccessful. Only a few calli were induced from proto-

corms (Table 2), indicating that either the composition of

the medium was not suitable for Paphiopedilum spp. or the

methodology was not suitable, since brown exudates cov-

ered most of the solid medium during explant culture and

may have affected the capacity of explants to absorb var-

ious nutrients. Lin et al. (2000) also found the inducement

of callus from protocorms of a Paphiopedilum hybrid

[P. callosum (Rchb.f.) Stein ‘Oakhil’ 9 P. lawrenceanum

(Rchb.f.) Pfitzer ‘Tradition’] to be extremely slow, but

about 42–65% of the protocorms in their experiment pro-

duced yellowish-white calli; however, stem, root tip, and

green leaf explants of another Paphiopedilum hybrid

[P. henryanum Braem ‘#1’ 9 P. philippinense (Rchb.f.)

Stein ‘#10’] failed to produce viable callus. The frequency

of explants forming callus of a Cymbidium hybrid was 50%

(Huan et al. 2004). These results imply that callus induc-

tion for interspecific hybrids is challenging and show that

the induction success for hybrids was greater than that for

the natural species since the PRG treatment combinations

in our investigation were relatively ineffective in inducing

Paphiopedilum callus (Table 2). Thus, methods and

materials other than those used in our study need to be

developed for the mass propagation of these orchid species.

Shoot multiplication in vitro

Nhut et al. (2007), working with Paphiopedilum delenatii,

showed that TDZ was more effective than BA in inducing

shoot organogenesis, even though the regeneration rate for

TDZ was only 75%. Our investigation revealed no advan-

tages of replacing BA with TDZ in terms of inducing shoot

organogenesis in our four Paphiopedilum species (Table 3).

Huang et al. (2001) found that the shoot number of Pa-

phiopedilum hybrids doubled every 12 weeks when treated

with BA and NAA, and that TDZ inhibited shoot prolifer-

ation and rooting of Paphiopedilum hybrids. Yan et al.

(2006) and Lu et al. (2001) reported that as many as two to

seven shoots per plant were produced on Cypripedium fla-

vum P. H. Hunt. et Summerh and Cymbidium ensifolium cv.

Yuh Hwa rhizomes when the latter were treated with BA.

Twelve shoots per node were induced for Vanda spathulata

(L.) Spreng. when treated with 44.4 lM BA with 17.1 lM

or 28.5 lM indole-3-acetic acid (IAA; William et al. 2003);

Dendrobium candidum Wall. ex Lindl. produced five shoots

per explant when treated with half-strength MS supple-

mented with 2 mg l-1 BA, 0.2 mg l-1 NAA, and

0.1 mg l-1 IBA (Luo et al. 2006). The maximum number of

adventitious buds per explants that has been reported to

date—an average of 24.5—were generated from Dendro-

bium candidum explants treated with 1.2 mg l-1 BA and

1.2 mg l-1 NAA (Zhao et al. 2007). Results from Martin

(2003), Chen et al. (2004a), Thomas and Michael (2007),

and Hong et al. (2008) show that, in most cases, Paphio-

pedilum spp. induce fewer shoots than other orchids.

The concentration of exogenous PGRs required for the

micropropagation of Paphiopedilum was found to be spe-

cies-specific (Table 3). A relatively high BA concentration

increased the shoot number in P. villosum var. densissimum

and P. armeniacum but decreased it in P. insigne. This

finding is similar to those of Molina et al. (2007) working

with Troyer citrange (Citrus sinensis [L.] Osbeck 9 Pon-

cirus trifoliata [L.] Raf.). In our study, a BA concentration

[3.5 mg l-1 caused extensive necrosis of P. insigne

explants. For P. bellatulum, organogenesis occurred with

only four PGR combinations. Shoot numbers in all four

orchid species were the same at TDZ concentrations

ranging from 0.01 to 0.1 mg l-1.

Organogenesis occurred on P. armeniacum rhizomes for

all PGR combinations (Table 3). Underground rhizomes of

temperate terrestrial Cymbidium species are useful organs

for micropropagation (Shimasaki and Uemoto 1990), but

this type of morphogenetic development is not a common

phenomenon in the Orchidaceae. There are 18 Chinese

species of Paphiopedilum, and only P. armeniacum and

P. micranthum have underground rhizomes, although a few

short rhizomes are occasionally observed on P. malipoense

S. C. Chen et Z. H. Tsi (Chen and Tsi 2003). Thus, prop-

agation of rhizomatous species, such as P. armeniacum,

facilitates clonal reproduction and enables mass propaga-

tion, whereas this is not true for the other three species used

in this study.

We found that when the BA concentration C2 mg l-1 or

higher, root tubercles were formed for P. insigne (Table 3,

Fig. 4j) and that the size of the tubercle and the fate of

plantlets depended on BA concentration. Root tubercles are

160 Plant Cell Tiss Organ Cult (2010) 101:151–162

123

atypical of Paphiopedilum spp. roots. P. insigne plantlets

turned brown when the concentration of BA was

[3.5 mg l-1. Thus, the concentration of the PGR is an

important factor to be taken into consideration in Paphio-

pedilum in vitro culture.

In most cases, a high ratio of cytokinin to auxin enhanced

shoot formation (PGR experiment, Table 3). This effect

was species-specific. Maximum shoot production for

P. villosum var. densissimum, P. insigne, P. bellatulum, and

P. armeniacum occurred at approximate ratios of 10:1, 2:1,

11:1, and 40:1 or 8:1, respectively. In some cases, such as

P. armeniacum, BA was unnecessary for shoot production;

in other cases, there was a wide range of ratios at which

maximum shoot formation occurred (e.g., P. armeniacum).

Shoot proliferation from leaf tissue is common in ferns

and dicotyledons and less common in monocotyledons

(Xiong and Wu 2003). The results from our investigation

indicate that the frequency of in vitro seed germination and

shoot organogenesis in Paphiopedilum species is lower

than that of most other orchid species, even when in vitro

techniques are used. However, our work sheds light on

techniques to maximize propagation success by employing

in vitro seed germination and shoot organogenesis meth-

odologies. As such, our results are relevant to the perpet-

uation of endangered Paphiopedilum spp.

Conclusion

The results of this study are promising in terms of devel-

oping a propagation protocol for the mass production of

P. villosum var. densissimum, P. bellatulum, P. insigne,

and P. armeniacum. Conventional seed propagation tech-

niques for Paphiopedilum spp. are relatively simple, but

the approximately 4-year period from seedling to seed

production renders seeds as an unlikely propagation start-

ing point, especially in terms of meeting horticultural

market demands. Thus, the use of plantlets or rhizomes

derived from in vitro seed germination for shoot multipli-

cation will significantly shorten the production cycle. Our

methods may prove to be of valuable for both horticultural

and conservation purposes.

Acknowledgments The authors thank Chunyan Han, Guangwan

Hu, and Yuan Liu for their kind help. This work was supported by the

Natural Science Foundation of Yunnan (2005C0053 M), the Ministry

of Science and Technology of China (2005DK21006) and the Min-

istry of Education of China (B08044 & MUC-985-3-3).

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